Rat Spinal Cord Cells Research Articles

A Dual Strategy-In Vitro and In Silico-To Evaluate Human Antitetanus mAbs Addressing Their Potential Protective Action on TeNT Endocytosis in Primary Rat Neuronal Cells.

Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.

Cleaved caspase-3 is present in the majority of glial cells in the intact rat spinal cord during postnatal life

Cell death is an essential process that occurs during the development of the central nervous system. Despite the availability of a wide range of commercially produced antibodies against various apoptotic markers, data regarding apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. We investigated apoptosis in rat spinal cord at different stages of ontogenesis (postnatal days 8, 29, and 90). For this purpose, we applied immunofluorescent detection of two widely used apoptotic markers, cleaved caspase-3 (cC3) and cleaved poly(ADP-ribose) polymerase (cPARP). Surprisingly, we found significant discrepancy between the number of cC3+ cells and PARP+ cells, with a ratio between 500:1 and 5000:1 in rat spinal cord at all postnatal time points. The majority of cC3+ cells were glial cells and did not exhibit an apoptotic phenotype. In contrast with in vivo results, in vitro analysis of primary cell cultures derived from neonatal rat spinal cord and treated with the apoptotic inductor staurosporine revealed a similar onset of occurrence of both cC3 and cPARP in cells subjected to apoptosis. Gene expression analysis of spinal cord revealed elevated expression of the Birc4 (XIAP), Birc2, and Birc5 (Survivin) genes, which are known potent inhibitors of apoptosis. Our data indicate that cC3 is not an exclusive marker of apoptosis, especially in glial cells, owing its possible presence in inhibited forms and/or its participation in other non-apoptotic roles. Therefore, cPARP appears to be a more appropriate marker to detect apoptosis.

The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material's surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment.

Electroacupuncture alleviates neuropathic pain caused by SNL by promoting M2 microglia polarization through PD-L1

As a common clinical disease, neuropathic pain is difficult to be cured with drugs. The occurrence and progression of pain is closely related to the response of spinal microglia. Aspartof the regulation of microglialactivity,PD-L1 playsacriticalrole. Loss of PD-L1 promoted the polarization of M1-like microglia. Increased expression of PD-L1 promoted M2-like polarization. Electroacupuncture has a significant analgesic effect in clinical practice, but its specific mechanism remains to be further explored. In this study, we verified the role of PD-L1 in EA analgesia and the underlying molecular mechanism through spinal nerve ligation (SNL) in rats and lipopolysaccharide (LPS)-treated BV2 microglial cells. Forbehavioralstudiesofrats,mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured, and spinal cord neuros were examined under transmission electron microscopyto determine changes to their myelin structure. The expression levels of PD-L1 and M1/M2-specific markers in rat spinal cord and BV2 microglial cells were measured by enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence staining and Western blot analysis. Our study showed that EA increased the pain threshold, reduced the destruction of myelin structure, promoted the expression of PD-L1 and PD-1, inhibited the MAPK signaling pathway, and promoted the conversion of microglial polarization from the M1 phenotype to the M2 phenotype in SNL rats. PD-L1 knockdown reversed these effects of EA. In addition, PD-L1 knockdown activated the MAPK signaling pathway, promoted microglial polarization to the M1 phenotype, decreased the expression of anti-inflammatory mediators and increased the expression of proinflammatory factors in LPS-stimulated BV2 microglial cells. Our results showed that EA may regulate the excitability of primary afferent neurons through PD-L1 and then inhibit the MAPK signaling pathway to promote the transformation of activated M1 microglia into M2 microglia, reduce inflammatory reactions, and finally achieve analgesic effects. A therapy targeting PD-L1 may be an effective strategy for treating neuropathic pain.

Thiadiazine-thione derivatives ameliorate STZ-induced diabetic neuropathy by regulating insulin and neuroinflammatory signaling.

Diabetes Mellitus is accompanied by chronic hyperglycemia, inflammation, and related molecular processes, which leads to diabetic neuropathy. In this work, we tested Thiadiazine-thione (TDT) synthetic derivatives TDT1 and TDT2 against streptozotocin (STZ)-induced diabetic neuropathy. Sprague Dawley's rats, SH-SY5Y neuronal and BV2 microglial cells were employed in this work, followed by behavioral, biochemical, and morphological studies utilizing RT-qPCR, ELISA, Immunoblotting, immunohistochemistry, Immunofluorescence, and in silico analyses. TDT1 and TDT2 abolished STZ-induced allodynia and hyperalgesia. Next, we examined IRS1/PI3K/AKT signaling to assess TDT1 and TDT2's impact on diabetic neuropathy. STZ downregulated IRS1, PI3K, AKT mRNA and protein expression in rat spinal cord and SH-SY5Y neuronal cells. TDT1 and TDT2 improved IRS1, PI3k, and AKT mRNA and protein expression. STZ elevated GSK3β mRNA and protein expression in vivo and in vitro, whereas TDT1 and TDT2 mitigated it. STZ increased the expression of inflammatory mediators such as p-NF-κB, TNF-α, and COX-2 in rat spinal cord lysates. TDT1 and TDT2 co-treatment with STZ decreased inflammatory cytokine expression by ameliorating astrocytosis (revealed by increased GFAP) and microgliosis (indicated by increased Iba1). TDT1 and TDT2 reduced STZ-induced JNK, Iba1, and COX-2 upregulation in BV2 microglial cells validating our in vivo findings. In silico molecular docking and MD simulations analyses suggested that TDT1 and TDT2 have IRS binding affinity, however, both compounds had an identical binding affinity, but distinct interaction pattern with IRS protein residues. Overall, these findings demonstrate that TDT derivatives mitigated STZ-induced neuropathy through modulating the insulin and inflammatory signaling pathways.

Stress-induced phosphoprotein 1 restrains spinal cord ischaemia-reperfusion injury by modulating NF-κB signalling.

Spinal cord injury (SCI), a major cause of disability, causes high global disease and economic burdens. Stress‐induced phosphoprotein 1 (STIP1) has been identified to be involved in spinal cord ischaemia‐reperfusion injury (SCII); however, the effect of STIP1 on SCII remains unclear until now. This study aimed to examine the role of STIP1 in SCII and unravel the possible mechanisms. Western blotting and immunohistochemical staining showed that STIP1 expression rapidly increased and then decreased in rat spinal cord following SCII treatment. Neurological function scoring, HE staining, immunohistochemical staining and Western blotting revealed that STIP1 overexpression alleviated SCII‐induced motor dysfunction of hind limbs, neuronal loss and inflammation in spinal cord, and inhibited activity of nuclear factor kappa B (NF‐κB) signalling in rats. Immunoprecipitation identified that STIP1 was co‐located with Iba‐1. In addition, STIP1 was found to ameliorate oxygen and glucose deprivation (OGD)‐induced inflammation and activation of NF‐κB signalling in mouse microglia BV2 cells, and STIP1 resulted in decrease of heat shock protein family A member 8 (HSPA8), increase of IκBβ expression and reduced binding of IκBβ to HSPA8 in BV2 cells. The results of the present study demonstrate that STIP1 alleviates ischaemia/reperfusion‐induced neuronal injury and inflammation in rat spinal cord and mouse microglial cells by deactivating NF‐κB signalling. These findings may provide novel insights for the clinical diagnosis and treatment of SCI.

MicroRNA-488 inhibits neural inflammation and apoptosis in spinal cord injury through restraint on the HMGB1/TLR4/NF-κB signaling pathway.

Secondary spinal cord injury (SCI), a reversible pathological change, involves neural inflammation and apoptosis. This study explored how microRNA (miR)-488, an inflammatory regulator as reported affected secondary SCI. In vivo, Wistar rats were clipped on the spinal cord for SCI induction. In vitro, PC-12 cells were treated with lipopolysaccharide (LPS) to induce cell injuries to mimic the environment during the secondary SCI. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry. The levels of inflammation-related factors (interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α) in the serum and PC-12 cells were determined by ELISA. The expressions of miR-488, high mobility group box 1 (HMGB1), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, toll-like receptor 4 (TLR4), phosphorylated (p)-p65 and total-p65 in rat spinal cord or PC-12 cells were analyzed by quantitative reverse transcription PCR or western blot. After SCI induction, rats exhibited low Basso-Beattie-Bresnahan scores, promoted the release of inflammation-related factors and downregulated miR-488. LPS treatment decreased cell viability, enhanced apoptosis and downregulated miR-488. Upregulating miR-488 neutralized LPS-induced releases of inflammation-related factors and expressions of Bax and cleaved caspase-3 and counteracted LPS-induced inhibition on Bcl-2 expression. MiR-488 directly targeted HMGB1 and miR-488 mimic decreased LPS-induced HMGB1 expression. Overexpressing HMGB1 counteracted miR-488 mimic-induced decreases in the expressions of TLR4 and p-p65 and the ratio of p-p65 to Total-p65 in LPS-treated PC-12 cells. MiR-488 inhibited neural inflammation and apoptosis in SCI via its binding with HMGB1-mediated restraint on the TLR4/NF-κB signaling pathway.

Effect of Prenatal Topiramate Administration on Early Postnatal Expression of GFAP in Radial Glial Cells in Albino Rat Cerebellum and Spinal Cord

Background: Topiramate is known as a modern drug approved for the treatment of patients with partial seizures. Radial glial cells are bipolar-shaped cells in the developing central nervous system of vertebrates. It can differentiate into other neurons and astrocytes. GFAP, Glial fibrillary acidic protein, is a glial intermediate filament protein that is known as the most comprehensive marker for astroglial cells including the radial glia Aim of the work: There are few reports on topiramate administration during pregnancy. Therefore, this drug was chosen to investigate its effects, if any, especially the expression of GFAP in radial glia in the pups’ cerebellum and spinal cord. Material and Methods: Male and female rats were coupled in one cage for mating. On the next morning, mating was approved by the presence of sperms in the female vaginal swab, it was considered as Gestation Day zero:0. Twelve female pregnant rats were divided equally into two groups; control and topiramate treated. Topiramate drug was given to the treated group via orogastric tube from GD zero (0) till the time of delivery in a dose of 100mg/kg the control rats were administered tap water by orogastric tube. At the end of the experiment, the newly born pups (PN1) were weighed, anesthetized with Ketamine (60 mg/kg i.p.). The cerebellum and spinal cord were dissected and weighted. The specimens were fixed in Bouin’s solution then processed for preparation of H& E and GFAP immunostained sections. Results:In H& E-stained sections, the cerebellum in the control group was formed of subsequent layers, purkinje cells were arranged in one row, rounded or fusiform shape. GFAP positive Immunostained radial fibers (Bregman glia) were detected in granular and purkinje cell layers. The treated group showed that cells were disorganized and degenerated with focal areas of neuronal loss. Weak GFAP reaction was observed in Bregman glia in granular, purkinje and white matter cell layers. The lumbosacral segment of the spinal cord ventral horn showed large multipolar motor neurons with vesicular nucleus (“owl-eye” appearance), dendrites and axons were obvious. Glial cells with deeply stained nuclei were noticed. In the treated group, neurons appeared degenerated, vacuolated cytoplasm and pyknotic nuclei with focal areas of cellular loss. GFAP immunostained section in the control group showed an intense reaction in the radial fibers of the glia of the ventral funiculus, in contrast to the treated group which showed a faint reaction in both ventral funiculus and horn. Conclusion: it could be concluded that daily regular administration of topiramate during a childbearing period in females may result in a neurotoxic effect in the cerebellum and spinal cord. So, the administration of this drug should be under strict medical observation.

Interleukin-1 receptor antagonist ameliorates the pain hypersensitivity, spinal inflammation and oxidative stress induced by systemic lipopolysaccharide in neonatal rats

Perinatal inflammation-induced reduction in pain threshold may alter pain sensitivity to hyperalgesia or allodynia which may persist into adulthood. In this study, we investigated the anti-inflammatory protective effect of interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory cytokine, on systemic lipopolysaccharide (LPS)-induced spinal cord inflammation and oxidative stress, thermal hyperalgesia, and mechanical allodynia in neonatal rats. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) or sterile saline was performed in postnatal day 5 (P5) rat pups, and IL-1ra (100 mg/kg) or saline was administered (i.p.) 5 min after LPS injection. Pain reflex behavior, spinal cord inflammation and oxidative stress were examined 24 h after LPS administration. Systemic LPS exposure led to a reduction of tactile threshold in the von Frey filament tests (mechanical allodynia) and pain response latency in the tail-flick test (thermal hyperalgesia) of P6 neonatal rats. Spinal cord inflammation was indicated by the increased numbers of activated glial cells including microglia (Iba1+) and astrocytes (GFAP+), and elevated levels of pro-inflammatory cytokine interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) 24 h after LPS treatment. LPS treatment induced spinal oxidative stress as evidenced by the increase in thiobarbituric acid reactive substances (TBARS) content in the spinal cord. LPS exposure also led to a significant increase in oligodendrocyte lineage population (Olig2+) and mature oligodendrocyte cells (APC+) in the neonatal rat spinal cord. IL-1ra treatment significantly reduced LPS-induced effects including hyperalgesia, allodynia, the increased number of activated microglia, astrocytes and oligodendrocytes, and elevated levels of IL-1β, COX-2, PGE2, and lipid peroxidation (TBARS) in the neonatal rat spinal cord. These data suggest that IL-1ra provides a protective effect against the development of pain hypersensitivity, spinal cord inflammation and oxidative stress in the neonatal rats following LPS exposure, which may be associated with the blockade of LPS-induced pro-inflammatory cytokine IL-1β.

Intracellular signaling similarity reveals neural stem cell-like properties of ependymal cells in the adult rat spinal cord.

Proliferation of ependymal cells of the adult spinal cord (SCEp cells) in the intact condition has been considered as a quite rare event. To visualize proliferating/proliferated SCEp cells, we used the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method to find that about two cells in the ependymal layer incorporated BrdU in a 10-μm-thick section. Because these two cells were not considered to undergo further proliferation, we analyzed the positioning and motility of two neighboring BrdU-incorporated proliferated cells and elucidated the tendency of the movement of SCEp cells to the outer side inside the ependymal layer. Even if it was rare, one of the proliferated cells in the ependymal layer differentiated into astrocytes. Gene introduction of Notch intracellular domain (NICD), a constitutively active form of Notch1, into SCEp cells demonstrated both increase in proliferation and induction of differentiation into astrocytes. Overexpression of Sox2 promoted proliferation in SCEp cells. The reaction of gene introduction of NICD and Sox2 indicates the similarity of intracellular signaling between SCEp cells and neural stem cells. Also, considering the fact that SCEp cells express these two factors in the intact condition, Notch and Sox2 are important for the cell fate control of SCEp cells in the intact condition.

Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord

The functional multipotency enables mesenchymal stem cells (MSCs) promising translational potentials in treating spinal cord injury (SCI). Yet the fate of MSCs grafted into the injured spinal cord has not been fully elucidated even in preclinical studies, rendering concerns of their safety and genuine efficacy. Here we used a rat spinal cord transection model to evaluate the cell fate of allograft bone marrow derived MSCs. With the application of immunosuppressant, donor cells, delivered by biocompatible scaffold, survived up to 8 weeks post-grafting. Discernible tubes formed by MSCs were observed beginning 2 weeks after transplantation and they dominated the morphological features of implanted MSCs at 8 weeks post-grafting. The results of immunocytochemistry and transmission electron microscopy displayed the formation of perineurium-like sheath by donor cells, which, in a manner comparable to the perineurium in peripheral nerve, enwrapped host myelins and axons. The MSC-derived perineurium-like sheath secreted a group of trophic factors and permissive extracellular matrix, and served as a physical and chemical barrier to insulate the inner nerve fibers from ambient oxidative insults by the secretion of soluble antioxidant, superoxide dismutase-3 (SOD3). As a result, many intact regenerating axons were preserved in the injury/graft site following the forming of perineurium-like sheath. A parallel study utilizing a good manufacturing practice (GMP) grade human umbilical cord-derived MSCs or allogenic MSCs in an acute contusive/compressive SCI model exhibited a similar perineurium-like sheath formed by surviving donor cells in rat spinal cord at 3 weeks post-grafting. The present study for the first time provides an unambiguous morphological evidence of perineurium-like sheath formed by transplanted MSCs and a novel therapeutic mechanism of MSCs in treating SCI.

Cyclosporine-immunosuppression does not affect survival of transplanted skin-derived precursor Schwann cells in the injured rat spinal cord.

A major goal of Schwann cell (SC) transplantation for spinal cord injury (SCI) is to fill the injury site to create a bridge for regenerating axons. However, transplantation of peripheral nerve SCs requires an invasive biopsy, which may result in nerve damage and donor site morbidity. SCs derived from multipotent stem cells found in skin dermis (SKP-SCs) are a promising alternative. Regardless of source, loss of grafted SCs post-grafting is an issue in studies of regeneration, with survival rates ranging from ∼1 to 20% after ≥6 weeks in rodent models of SCI. Immune rejection has been implicated in these low survival rates. Therefore, our aim was to explore the role of the immune response on grafted SKP-SC survival in Fischer rats with a spinal hemisection injury. We compared SKP-SC survival 6 weeks post-transplantation in: (I) cyclosporine-immunosuppressed rats (n=8), (II) immunocompetent rats (n=9), and (III) rats of a different sub-strain than the SKP-SC donor rats (n=7). SKP-SC survival was similar in all groups, suggesting immune rejection was not a main factor in SKP-SC loss observed in this study. SKP-SCs were consistently found on laminin expressed at the injury site, indicating detachment-mediated apoptosis (i.e., anoikis) might play a major role in grafted cell loss.

Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury

Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury.

GAP-43 Protein and Its Proteolytic Fragment in Spinal Cord Cells in Rats with Experimental Allergic Encephalomyelitis

The limited regenerative capacity of the central nervous system (CNS) is one of the key factors promoting the development of neurodegenerative diseases, including multiple sclerosis. The regenerative potential of CNS cells can be evaluated in terms of the level of GAP-43 protein, which is involved in the processes of neuritogenesis and growth cone navigation. Cleavage of GAP-43 by m-calpain to form the fragment GAP-43-3, which lacks 40 N-terminal amino acids, probably leads to loss of the initial functions of the protein, which may have adverse influences on the regenerative potential of the CNS. The present study was performed on the most appropriate model of multiple sclerosis – experimental allergic encephalomyelitis (EAE) in rats. The spinal cord of animals with EAE was shown to have inflammatory infiltrates and deformed motoneurons; limb pareses and paralysis developed. When EAE was mild, about 60% of GAP-43 in rat spinal cord cells was present in the form of GAP-43-3. In severe forms of EAE, with tetraplegia, the extent of GAP-43 proteolysis by calpain reached 85%. In this situation, GAP-43-3 was found mainly in microglial cells. We suggest that during the development of EAE, proteolytic cleavage of GAP-43 protein by m-calpain to form GAP-43-3 can significantly alter its properties, suppressing the regenerative ability of CNS cells and exacerbating the course of the pathological process.

The expression of PLP/DM-20 mRNA is restricted to the oligodendrocyte-lineage cells in the adult rat spinal cord.

Proteolipid protein (PLP) is the major component of myelin; its gene encodes two major splicing variants: PLP and DM-20. Compared with PLP, DM-20 lacks the amino acids encoded by exon IIIb. The expression of PLP/DM-20 in cells outside the oligodendrocyte-lineage is unclear. To address this issue, we analyzed the detailed expression pattern of PLP/DM-20 mRNA in the adult rat spinal cord by in situ hybridization (ISH) with a cRNA probe complementary to DM-20 mRNA, which has been used to detect both PLP and DM-20 both mRNA. ISH did not label the cells expressing NeuN nor glial fibrillary acidic protein but detected those expressing Olig2, indicating that PLP/DM-20 mRNA are expressed only in oligodendrocyte-lineage cells. This cell population was expected to contain NG2-expressing oligodendrocyte precursor cells (OPCs), because some exhibited the expression of glutathione S-transferase pi isoform in the nucleus. A recent publication showed that OPCs express PLP but not DM-20 mRNA. However, no OPCs were detected. We performed ISH with a cRNA probe that specifically recognizes PLP mRNA to successfully detect some OPCs. Additionally, OPCs were detected by ISH with a cRNA probe complementary to DM-20 mRNA that was digested via alkaline hydrolysis prior to ISH. These findings collectively demonstrate that PLP and DM-20 mRNA expression is restricted to oligodendrocyte-lineage cells, and imply that the undigested cRNA probe complementary to the full-length DM-20 mRNA sequence only recognizes DM-20 mRNA and not the PLP counterpart when applied to ISH without denaturation/digestion methods.

Diacerein reduces joint damage, pain behavior and inhibits transient receptor potential vanilloid 1, matrix metalloproteinase and glial cells in rat spinal cord.

To investigate the antinociceptive, antiedematogenic and chondroprotective effects of diacerein (DIA) in a model of joint inflammation induced by complete Freund's adjuvant (CFA), as well as to investigate the involvement of metalloproteinase (MMP)-9, transient receptor potential vanilloid 1 (TRPV1) and glial cells in DIA's action mechanism. Complete Freund's adjuvant was injected into the knee joint of male rats. We observed mechanical and cold hypersensitivity, vocalization and spontaneous pain-related behaviors, as well as edema of the knee. Tissue samples of the knee were stained with Cason`s technique and the thickness of the condilus cartilage was measured. Immunohistochemical analysis was performed on the spinal cord using anti-GFAP (glial fibrillary acidic protein), anti-MMP and anti-TRPV1 antibodies. Sections of the dorsal horns of the spinal cord were captured and an optical density was obtained. Complete Freund's adjuvant induced mechanical and thermal hypersensitivity, as well as joint edema and changes in the synovial membrane and cartilage. DIA (30 mg/kg, orally, daily) significantly inhibited mechanical (58 ± 10-87 ± 3%) and thermal (66 ± 12-87 ± 8%) hypersensitivity, vocalization (83 ± 5-41 ± 11%), spontaneous pain score, joint swelling (60 ± 6-40 ± 9%), as well as the histological changes induced by CFA. In addition, DIA inhibited astrocyte activation, and prevented the increase of MMP-9 and TRPV1 expression in the spinal cord of the animals subjected to CFA injections. In short, this study shows that DIA reduces joint damage and hypersensitivity associated with inflammation induced by CFA through the inhibition of astroglial activation and decreases the expression of TRPV1 and MMP-9 in the rat spinal cord.

Distribution and phenotype of TrkB oligodendrocyte lineage cells in the adult rat spinal cord

The distribution and phenotype of a previously undescribed population of nonneuronal cells in the intact spinal cord that expresses TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4), were characterized by examining the extent of co-localization of TrkB with NG2, which identifies oligodendrocyte progenitors (OPCs) or CC1, a marker for mature oligodendrocytes (OLs). All TrkB nonneuronal cells expressed Olig2, confirming their role in the OL lineage. Similar to OPCs and OLs, TrkB cells resided in gray and white matter of the spinal cord in similar abundance. Less than 2% of TrkB cells expressed NG2, while over 80% of TrkB cells in the adult spinal cord co-expressed CC1. Most OPCs did not express detectable levels of TrkB, however a small OPC pool (~5%) showed TrkB immunoreactivity. The majority of mature OLs (~65%) expressed TrkB, but a population of mature OLs (~36%) did not express TrkB at detectable levels, and 17% of TrkB nonneuronal cells did not express NG2 or CC1. Approximately 20% of the TrkB nonneuronal population in the ventral horn resided in close proximity to motor neurons and were categorized as perineuronal. We conclude that TrkB is expressed by several pools of OL lineage cells in the adult spinal cord. These findings are important in understanding the neurotrophin regulation of OL lineage cells in the adult spinal cord.

Persistence of Botulinum Neurotoxin A Subtypes 1-5 in Primary Rat Spinal Cord Cells

Botulinum neurotoxins (BoNTs) are the most poisonous substances known and cause the severe disease botulism. BoNTs have also been remarkably effective as therapeutics in treating many neuronal and neuromuscular disorders. One of the hallmarks of BoNTs, particularly serotype A, is its long persistence of 2-6 months in patients at concentrations as low as fM or pM. The mechanisms for this persistence are currently unclear. In this study we determined the persistence of the BoNT/A subtypes 1 through 5 in primary rat spinal neurons. Remarkably, the duration of intracellular enzymatic activity of BoNT/A1, /A2, /A4 and /A5 was shown to be at least 10 months. Conversely, the effects of BoNT/A3 were observed for up to ∼5 months. An intermittent dosing with BoNT/E showed intracellular activity of the shorter acting BoNT/E for 2–3 weeks, followed by reoccurrence and persistence of BoNT/A-induced SNAP-25 cleavage products.

Temporal distribution of p300/CBP immunoreactivity in the adult rat spinal dorsal horn following chronic constriction injury (CCI).

p300 and its homolog cyclic AMP response element binding protein (CBP) are coactivators that were identified to participate in many biological processes including neural development and cognition. Their roles within the rodent spinal cord have not been reported systematically; in this study, their spatiotemporal distribution in the spinal cord of adult rat following chronic constriction injury (CCI) was studied. p300 and CBP expressed predominantly in nuclei in the gray matter of rat spinal cord. Rats undergoing CCI surgery showed increased p300/CBP immunoreactivity (IR) compared with normal control and sham-operated rats. The number of IR cells reached the peak at day 14 following CCI compared with those on day 3, 7, and 21, accompanied with significant behavioral changes of neuropathic pain. Cell-type determination by immunofluorescence at day 14 following CCI revealed that p300 and CBP expressed in neurons, but not in astrocytes or microglial cells. These results suggest that p300 and CBP are probably involved in the maintenance of neuropathic pain on spinal cord level. Furthermore, p300 and CBP may serve as a sensor only in neurons but not in astrocytes or microglia cells in the adult rat spinal cord.

Novel Application of Human Neurons Derived from Induced Pluripotent Stem Cells for Highly Sensitive Botulinum Neurotoxin Detection

Human induced pluripotent stem cells (hiPSC) hold great promise for providing various differentiated cell models for in vitro toxigenicity testing. For Clostridium botulinum neurotoxin (BoNT) detection and mechanistic studies, several cell models currently exist, but none examine toxin function with species-specific relevance while exhibiting high sensitivity. The most sensitive cell models to date are mouse or rat primary cells and neurons derived from mouse embryonic stem cells, both of which require significant technical expertise for culture preparation. This study describes for the first time the use of hiPSC-derived neurons for BoNT detection. The neurons used in this study were differentiated and cryopreserved by Cellular Dynamics International (Madison, WI) and consist of an almost pure pan-neuronal population of predominantly gamma aminoisobutyric acidergic and glutamatergic neurons. Western blot and quantitative PCR data show that these neurons express all the necessary receptors and substrates for BoNT intoxication. BoNT/A intoxication studies demonstrate that the hiPSC-derived neurons reproducibly and quantitatively detect biologically active BoNT/A with high sensitivity (EC(50) ∼0.3 U). Additionally, the quantitative detection of BoNT serotypes B, C, E, and BoNT/A complex was demonstrated, and BoNT/A specificity was confirmed through antibody protection studies. A direct comparison of BoNT detection using primary rat spinal cord cells and hiPSC-derived neurons showed equal or increased sensitivity, a steeper dose-response curve and a more complete SNARE protein target cleavage for hiPSC-derived neurons. In summary, these data suggest that neurons derived from hiPSCs provide an ideal and highly sensitive platform for BoNT potency determination, neutralizing antibody detection and for mechanistic studies.